Efficacy of Application of Plasma Rich in Growth Factors Along with the Tunnel Technique for Treatment of Gingival Recession: a Clinical Trial

Statement of the Problem: The tunnel technique has shown promising results in treatment of gingival recession. Plasma rich in growth factors (PRGF) is considered effective for soft tissue regeneration since it is a rich source of growth factors. Purpose: This clinical trial aimed to assess the efficacy of PRGF along with the tunnel technique and connective tissue graft for treatment of gingival recession. Materials and Method: In this controlled clinical trial, 20 areas around anterior and posterior teeth in 3 patients with gingival recession were bilaterally selected. The tunnel technique was used with and without PRGF in the test and control groups, respectively (10 areas in each group). The keratinized gingival width (KGW), clinical attachment level (CAL), clinical probing depth (PD), cementoenamel junction (CEJ) to mucogingival junction (MGJ) distance, and the esthetic visual analog scale (EVAS) score were evaluated preoperatively and at 6 weeks and 3 months, postoperatively. The gingival recession width (RW) and vertical recession depth (VRD) were assessed preoperatively and at 2 weeks and 3 months, postoperatively. The pain visual analog scale (PVAS) score was measured at 1, 3 and 7 days, post-treatment and the healing index (HI) was measured at 1, 3 and 7 days and 1 month, postoperatively. The root coverage percentage was assessed during 3 months. Paired t-test and repeated measures ANOVA were used for statistical analyses. p Value< 0.05 was considered statistically significant. Results: Significant improvements were noted in all tested parameters in both groups (p< 0.05). The mean root coverage percentage after 6 months was 88.68%±20.69% and 78.77%±24.94% in the test and control groups, respectively. None of the tested parameters were significantly different between two groups (p> 0.05). Conclusion: Treatment of gingival recession with the tunnel technique can yield favorable clinical outcome, irrespective of the employment of PRGF.


Introduction
Gingival recession is defined as apical dislodgment of the gingival margin relative to the cementoenamel junction (CEJ) due to the loss of periodontal attachments, and is currently a common clinical finding [1][2]. The etiology of gingival recession includes periodontal disease, poor oral hygiene, frenulum stretching, and bone dehiscence, inappropriate path of eruption of tooth, tooth malposition, gingival viral infections, and subgingival plaque accumulation [3]. In addition, toothbrushing trauma, especially in young individuals, can play a role in progression of gingival recession [4]. Gingival recession can compromise smile esthetics and affect the mastication function as well [5][6]. Also, it complicates plaque control [4]. Due to the complications of gingival recession such as tooth hypersensitivity [7], root caries [8], esthetic concerns and decreased attached gingiva, periodontists have long been in search of novel techniques for proper management of this condition. Treatment of gingival recession is highly challenging for dental clinicians since an ideal treatment for gingival recession should be able to restore the lost anatomy of the mucogingival junction (MGJ), improve the esthetic appearance of the tooth, regenerate or restore cementum, induce reattachment of periodontal fibers and generation of the supporting bone, eliminate tooth hyper-sensitivity and prevent root caries. To date, several surgical techniques such as laterally positioned flap [9], coronally positioned flap [10], free gingival graft [11][12] and sub-epithelial connective tissue graft with coronally positioned flap [13] have been introduced and evaluated for treatment of gingival recession. However, application of coronally advanced flap along with connective tissue graft (bilaminar technique) currently serves as the gold standard for this purpose [14]. Since adequate keratinized gingiva is not available at the recession site, soft tissue grafts are obtained from an intraoral donor site. The use of connective tissue for root surface coverage was first suggested by Langer and Calagna [15] in 1982. In graft procedures, the main challenge is to preserve the blood supply of the recipient's site to avoid necrosis and defects.
Palatal mucosa can serve as an appropriate graft donor site due to its keratinized tissue [15][16].
Therefore, an intraoral autogenous graft with proper blood supply can be helpful for treatment of gingival recessions. The tunnel technique as a one-step surgical procedure can be used for treatment of Miller's class 1 and 2 multiple gingival recessions [16]. The length of the hard palate and the thickness of the palatal mucosa are important factors to consider in selection of this technique. This technique has many advantages. It preserves the integrity of the papilla, and the flap does not require a vertical releasing incision; thus, the connective tissue and periosteal hemorrhage is prevented. The surgery is performed with minimal trauma. Also, the grafted tissue is fixed and does not move in this technique and is also less exposed. The level of pain of patients is often minimal after this procedure and the healing peri-od is fast. Moreover, esthetic results are achieved sooner than other techniques with minimal scarring.
According to the protocol described by Allen [17], the tunnel technique requires a supra-periosteal mucosal flap with intra-sulcular incisions. By using this method, cervical gingival movement can be achieved by creating a space. Next, the interdental papilla is undermined and a mucogingival tunnel is created between the spaces.
After that, a connective tissue graft is placed inside the tunnel, with part of it covering the recessed gingiva. It is then sutured in a fixed position [4].
The use of endogenous and biologically active proteins for regenerative purposes has opened a new path for tissue regeneration. Growth factors are biological mediators that play a key role in proliferation, chemotaxis, and cell differentiation. They act through specific receptors located on the surface of the cells and guide the healing process. Growth factors are like hormones that are not released into the blood circulation and only act locally; some growth factors can cause premature changes in the G0 to G1 phases of cell division, and even have the ability to stimulate DNA synthesis in some certain cells [12].
In 1999, Anitua [18] described a new technique for preparation of plasma-rich platelets known as plasma rich in growth factors (PRGF). PRGF is prepared autogenously and is rich in biological mediators that accelerate hard and soft tissue regeneration. Plasma-derived adhesion molecules such as fibrinogen, fibronectin, vitronectin and thrombospondin-1 act as a matrix or scaffold and attract precursor cells and platelets. Platelets are a rich source of growth factors such as platelet-derived growth factor, transforming growth factor beta, vascular endothelial growth factor, fibroblast growth factor, insulin-like growth factor and granulocytemacrophage colony-stimulating factor [19]. KGW was characterized as the distance between the free gingival margin and the MGJ. The CAL was defined as the distance from the CEJ to the bottom of the gingival margin at the midpoint of the buccal gingival margin. PD was the distance between the free gingival margin and the bottom of the gingival sulcus at the midpoint of the buccal gingival margin [20]. VRD was the distance between the CEJ and the free gingival margin (at the midpoint of the buccal surface). The RW was defined as the width of recession at 1 mm apical to the CEJ in the mesiodistal dimension. The CEJ to MGJ distance was the distance between the CEJ and MGJ in the middle of the buccal surface of the tooth [21]. In order to assess the possible movement of the MGJ, CEJ was considered as a fixed reference line. The root coverage percentage was calculated based on the following equation: The HI was calculated according to the criteria by Landry [22]. The EVAS score was determined using a      In one side, the gingival recession was treated with the tunnel technique using PRGF (test group) while on the other side, gingival recession was treated with the tunnel technique without PRGF (control group). All surgical procedures were performed by one single sur-geon. In the preoperative phase, the clinical parameters were measured by an examiner who was blinded to the procedure and group allocation of sites. The measurements were made using a periodontal probe (Williams probe; HU-Friedy, Chicago, IL, USA) with 1mm accuracy. PRGF was prepared right before surgery as described by Anitua [23]. Before surgery, 20 mL of venous blood was collected from the patient and transferred into 5 mL tubes containing 3.8% sodium citrate as anticoagulant. Then, the tubes were centrifuged (En- and between-group differences were analyzed before and after treatment using t-test and paired t-test. Comparisons were carried out using repeated measures ANOVA (non-parametric test) whenever required. p< 0.05 was considered statistically significant.

Results
Both groups showed significant changes in KGW at 6 weeks (p< 0.001 in the test and p< 0.001 in the control group) and 3 months (p< 0.001 in the test and p< 0.001 in the control group) after treatment compared with baseline. The changes in CAL were also significant at 6 weeks (p< 0.001 in the test and p< 0.001 in the control group) and 3 months (p< 0.001 in the test and P < 0.001 in the control group) after surgery compared with baseline in both groups ( Table 1) Table 1 presents the results regarding PVAS score, HI, PD and EVAS score. All measured parameters showed significant improvement after treatment in both groups. But, none of the evaluated parameters were significantly different between the two groups (p> 0.05).  Huang et al. [28] used subepithelial connective tissue graft technique; while, in the present study and the study by Thalmair et al. [29] the tunnel technique was adopted to treat gingival recession. It seems that the tunnel technique is more predictable than the subepithelial connective tissue graft technique, which may be due to its lower invasiveness and higher blood supply. The baseline KGW value may be another reason for different findings of studies, as in the study by Jenabian et al. [21] the pre-treatment KGW was recorded to be 4.8mm and 4mm in the case and control groups, respectively, while in our study, this value was 3.6 mm. The same result was found in the study by Lafzi et al. [30].

No significant difference was noted in HI between
In the current study, the mean percentage of root co-  [21]. The findings of other studies such as those of Lafzi et al. [26], Jankovic et al. [27], and Cheung et al. [31] were similar to the results of Jenabian et al. [21]. However, in the present study, the mean percentage of root coverage was higher than that reported by previous studies [26][27]32]. Again, difference in technique of treatment seems to play a role in controversial results.
KGW is an important parameter when interpreting the outcome of treatment of gingival recession. It is also important in maintaining gingival health [21]. In the present study, the mean KGW in the experimental group increased by 1.75 mm and 1.6 mm at 6 weeks and 3 months after treatment, respectively. This value was 2.2 mm and 1.95 mm, respectively in the control group, and there was no statistically significant difference between the two groups. These values in the study by Jenabian et al. [21] were lower at 6 months (0.95 and 0.59 mm, respectively). Also, other studies indicated different magnitudes of improvement in KGW [28,31,[33][34].
The pain score in the present study was evaluated using the VAS. In general, the mean pain score in the test group was significantly lower than that in the control group. Also, in the study by Jankovic et al. [27] the severity of pain in the connective tissue graft group was significantly higher than that in platelet-rich plasma group with connective tissue graft. Moreover, in both groups, the level of pain significantly decreased in the first 7 days. In the study by Jenabian et al. [21], there was no significant difference in pain score between the two groups. Differences in pain outcome may be due to different surgical techniques, post-surgical medications, surgeons' skills, and the threshold of pain in patients.
In this study, we quantified the tissue repair using the HI by Landry [22]. The results showed that tissue repair increased from day 1 after treatment to 1 month after treatment, and recovery was achieved, but the difference in tissue repair between the two groups was not significant. In the study by Jenabian et al. [21], there was no significant difference in tissue repair between the experimental and control groups; however, in the study by Jankovic et al. [27] wound healing was better in the group with platelet-rich plasma than the group with connective tissue graft alone. This difference may be related to the different methodologies.
In the present study, the mean CAL significantly decreased in both groups (1.75mm in the test and 1.8mm in the control group) by up to 3 months after the treatment; but the difference in this regard was not significant between the study groups. This finding was similar to the results of Jenabian et al. [21], who showed that the mean CAL at 6 months after treatment significantly decreased in both groups (23.1 mm in the experimental group and 1 mm in the control group). However, the difference between the groups was not significant.
In this study, the RW significantly decreased in both groups (2.8 mm in the test and 3.25 mm in the control group after 3 months). However, the difference was not significant between the groups. In general, the mean distance between CEJ and MGJ increased at 3 months after treatment, but there was no significant difference between the two groups. Jenabian et al. [21] found similar results and did not report significant differences between the two groups either.
In this study, the esthetic index in both groups did